李芳仁臺灣大學：分子醫學研究所李純純Li, Chun-ChunChun-ChunLi2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51361腺嘌呤核苷二磷酸核醣化因子 (ADP-ribosylation factor, Arf) 是Ras蛋白GTPase家族的一支。在哺乳動物中已發現有六個Arf蛋白、二十二個腺嘌呤核苷二磷酸核醣化因子相似蛋白 (Arl) 和兩個Sar蛋白。Arfs和Arl1已知扮演著調控細胞內的囊泡運輸、磷脂酶的活化、參與磷脂三激酶的訊息調控等等的功能。但目前我們對於其他Arl蛋白的功能卻仍不清楚。在本論文研究中，我們將探討和發育調控有關之ARL4D蛋白的生物特性以及其功能。利用螢光染色法，我們發現在細胞分裂間期 (interphase) 時，ARL4D蛋白分布在細胞膜上和細胞核內，且ARL4D蛋白必須依賴和GTP鍵結及N端 (amino-terminal region) 的荳蔻酸化 (myristoylation) 修飾才可分布於細胞膜上。於細胞有絲分裂時則明顯可見ARL4D蛋白分佈於中心體 (centrosome) 區域。利用酵母菌雙雜合篩選法找出與ARL4D蛋白直接作用的蛋白質cytohesin-2/ARNO。cytohesin-2/ARNO是一個Arf的鳥糞嘌呤核苷酸轉換因子(guanine nucleotide exchange factor)，cytohesin-2/ARNO位在細胞膜上時可活化Arf6而能調控細胞中多種之生理功能‚如調控肌動蛋白(actin)的重組和細胞膜的摺縐(Radhakrishna et al.)。在本研究中，我們發現與GTP鍵結之ARL4D蛋白可與cytohesin-2/ARNO相互結合，此相互結合是透過cytohesin-2/ARNO羧基端的PH domain和polybasic c domain。與GTP鍵結之ARL4D可使cytohesin-2/ARNO由細胞質中分布到GTP-ARL4D集中之細胞膜。此外，我們還發現ARL4D蛋白具有和cytohesin-2/ARNO相似之功能，GTP-ARL4D可活化Arf6而影響細胞中肌動蛋白壓力纖維 (actin stress fibers) 之穩定，使肌動蛋白壓力纖維分解。表現沒有酵素催化能力的cytohesin-2/ARNO(E156K)突變或是使用RNA干擾 (siRNA) 技術調降cytohesin-2/ARNO蛋白表達後可抑制ARL4D所造成的肌動蛋白壓力纖維分解。而調降ARL4D蛋白表達則可抑制細胞移動。進一步，ARL4D蛋白造成之cytohesin-2/ARNO改變分布到細胞膜並不需要磷脂三激酶 (phosphoinositide 3-kinase) 的活化。總而言之‚我們的結果顯示ARL4D蛋白是一個新的cytohesin-2/ARNO的上游調控者而促進Arf6的活化和調節肌動蛋白的重組。另一方面與GDP鍵結之ARL4D (ARL4D(T35N)) 在細胞中，則有許多不同的分佈型式，包括座落於分布於分散於細胞質、粒線體和細胞核。使用粒線體膜電位染劑 (MitoTracker Red) 發現GDP-ARL4D會造成粒線體膜電位差消失，但細胞色素 (cytochrome c) 並未釋放於細胞質中且不會出現明顯細胞死亡的情形。因此，根據我們的研究發現，我們推測ARL4D在細胞中參與調控細胞骨架的重組和粒線體的功能等作用。ADP-ribosylation factor (Arf) family of small GTPase in mammals consists of 6 Arfs, 22 Arls (Arf-like), and 2 Sar proteins. The Arf family is best known for the role of Arfs and Arl1 as regulators for coat protein recruitment and phospholipid metabolism, but the biological functions of most Arls remain largely unknown. In this dissertation, a developmentally regulated member of Arl protein, ARL4D, was characterized. In interphase cells, ARL4D localizes to plasma membrane and nuclei and localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. In mitotic cells, ARL4D was associated predominantly with centrosomal region. We identified an ARL4D interacting protein, cytohesin-2/ARNO. Cytohesin-2/ARNO is a guanine nucleotide-exchange factor (GEF) for Arf and at the plasma membrane it can activate Arf6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or siRNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling. We also show that GTP-binding-defective mutant ARL4D(T35N) localizes to mitochondria and causes dissipation of mitochondrial membrane potential, but not induce release of cytochrome c. Based on our data, we infer that ARL4D might regulate different cellular processes, including cytoskeleton reorganization and mitochondrial function.Table of Contents 1 中文摘要 3 Abstract 4 Abbreviations 5 Introduction 7 Materials and Methods 28 Results 37 Ⅰ. Subcellular localization of ARL4D 37 Ⅱ. Identification and functional characterization of ARL4D-interacting proteins 41 Ⅱ-1. ARNO 42 Ⅱ-2. TACC3 52 Ⅱ-3. EB1 53 Ⅲ. To characterize the function of ARL4D on mitochondria 54 Discussion 58 Tables 68 Tables 68 Table 1. Antibodies used in this thesis 68 Table 1. Antibodies used in this thesis 68 Table 1. Antibodies used in this thesis (Continued) 69 Table 2. Oligonucleotides used in this thesis 70 Table 3. A brief summary of vectors used in this thesis 75 Table 4. Putative ARL4D interacting proteins 76 Table 5. Interactions between binding partners of ARL4D and ARL GTPases 77 Figures 78 Figure 1. Specificity of ARL4D antibody. 78 Figure 2. Endogenous ARL4D localized at the plasma membrane. 79 Figure 3. Localization of ARL4D at plasma membrane is GTP-dependent. 81 Figure 4. ARL4D localized around the centrosome during mitosis. 82 Figure 5. The localization of ARL4D(Q80L) and ARL4D(T35N) during mitosis. 83 Figure 6. The subcellular localization of GFP-ARL4D. 84 Figure 7. Endogenous ARL4D localized around the mitotic centrosome. 85 Figure 8. ARL4D is degraded rapidly by the proteasome. 86 Figure 9. ARL4D is a nuclear-cytoplasmic shuttling protein. 88 Figure 10. ARNO interacts with ARL4D. 89 Figure 11. The PH domain of ARNO interacts with ARL4D. 90 Figure 12. Effect of ARNO and ARNO(E156K) on GTPγS binding to ARL4D and ARF6. 91 Figure 13. ARL4D induces ARNO redistribution to plasma membrane protrusions and ruffles. 93 Figure 14. ARL4D(Q80L) promoted ARNO translocation. 94 Figure 15. ARL4D(Q80L) did not induce ARNOΔCT redistribute to the plasma membrane. 95 Figure 16. ARL4D(Q80L) induces redistribution of ARNO(E156K) to the plasma membrane. 96 Figure 17. ARL4D promotes activation of ARF6. 97 Figure 18. ARL4D(Q80L) induces actin stress fiber disassembly. 99 Figure 19. Requirement for ARL4D in cell migration. 100 Figure 20. ARL4D-induced translocation of ARNO is not dependent on PI3K signaling. 101 Figure 21. Active form of ARL4D induces redistribution of other members of the cytohesin family to the plasma membrane. 103 Figure 22. ARL4D did not recruit Akt-PH-GFP to the plasma membrane. 104 Figure 23. Active forms of ARL4A or ARL4C can recruit ARNO to the plasma membrane. 105 Figure 24. Interaction of ARL4D(Q80L) with TACC3 in a two-hybrid assay. 106 Figure 25. GFP-ARL4D partially colocalized with TACC3 at spindle poles apparatus. 107 Figure 26. GFP-ARL4D colocalized with EB1 at centrosome. 108 Figure 27. Enhanced stability of microtubule by ARL4D(Q80L). 109 Figure 28. GDP-bound ARL4D(T35N) localizes to mitochondria 110 Figure 29. Subcellular distribution of GTP-bound ARL4D(Q80L) or GDP-bound ARL4D(T35N) 111 Figure 30. C-terminal deletion ARL4D also localize to mitochondria and affect the mitochondrial morphology 112 Figure 31. ARL4(T35N) causes dissipation of mitochondrial membrane potential. 114 Figure 32. GDP-bound ARL4D didn’t cause cytochrome c release. 115 Figure 33. ARL4D translocate to mitochondria on oxidative stress. 116 Figure 34. Model for the role of ARL4D in recruitment of ARNO and activation of ARF6. 117 Figure S1. The structural GDP/GTP cycle of ARF6. 118 Figure S2. Regulation of ARF activity by GEFs that accelerate GTP binding and GAPs that activate GTP hydrolysis by ARF 119 Figure S3. Regulation of COPI-coat assembly and vesicle budding by ARF1. 120 Figure S4. ARF6 regulates clathrin-dependent and clathrin-independent endocytic pathways. 121 Figure S5. Classification and domain structures of Arf GAPs. 122 Figure S6. The localization of C-terminal myc tagged ARL4D. 123 Reference 1248439883 bytesapplication/pdfen-US腺嘌呤核苷二磷酸核糖化因子鳥糞嘌呤核苷酸結合蛋白細胞膜荳蔻酸化ADP-ribosylation factorGuanine nucleotide-binding proteinGTPaseplasma membranemyristoylationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51361/1/ntu-96-F89448007-1.pdf